Method of sulfating a tyrosine moiety

ABSTRACT

DISCLOSED HEREIN IS A METHOD OF SULFATING THE HYDROXYL GROUP OF AN L-TYROSINE MOIETY BY TREATING SAID MOIETY WITH A MIXTURE OF POTASSIUM BISULFATE AND CONCENTRATED SULFURIC ACID.

United States Patent Oflice METHOD OF SULFATING A TYROSINE MOIETY MiguelA. Ondetti, North Brunswick, Josip Pluscec, East Brunswick, and John T.Sheehan, Middlesex, N.J., and Johan E. Jorpes, Stockholm, and ViktorMutt, Farsta, Sweden, assignors to E. R. Squibb & Sons, Inc., New York,N.Y.

No Drawing. Original application May 3, 1968, Ser. No. 726,558. Dividedand this application Dec. 23, 1969, Ser. No. 889,770

Int. Cl. C07c 103/52 US. Cl. 260-1125 10 Claims ABSTRACT OF THEDISCLOSURE Disclosed herein is a method of sulfating the hydroxyl groupof an L-tyrosine moiety by treating said moiety with a mixture ofpotassium bisulfate and concentrated sulfuric acid.

RELATED APPLICATION 3,778,429 Patented Dec. 11, 1973 aspartyl 0 sulfateL tyrosyl, and to the intermediates and their salts employed in thepreparation thereof.

Peptide salts encompassed by the above include, for instance,hydrochlorides, hydrobrornides, acetates, fluoroacetates, such astrifluoroacetate, chloroacetates such as dichloroacetate, and the like,as well as ammonium salts such as dicyclohexylammonium,triethylammonium, morpholinium, pyridinium, and the like.

The final products of this invention are peptide amides containing aminoacid residues of L-isoleucine (Ileu), L-serine (Ser), L-arginine (Arg),L-tyrosine (Tyr), L- aspartic acid (Asp), L-methionine (Met), glycine(Gly), L-tryptophane (Trp) and L-phenylalanine (Phe).

These products have been found to possess cholecystokinin activity. Thatis, they have the ability to stimulate the contraction of the gallbladder. Thus, they find utility as diagnostic aids in X-ray examinationof the gall bladder in the same manner as cholecystokinin. For suchpurposes, they may be administered either intravenously orsubcutaneously to an animal species (e.g., cats or dogs)in a singledosage of about 0.0002 to 0.0003 mg./kg. of body weight.

For this purpose they may be administered parenterally by incorporatingthe appropriate dosage of the compound with carriers to form injectablesaccording to standard pharmaceutical practice.

The compounds of this invention may be prepared in accordance with thefollowing reaction scheme wherein B represents benzyl and X and Y are ashereinafter set forth:

I Met-Gly-Trp-Met-Asp-Phe-NH,

X-Asp-Tyr-NHNH, (V) 1 Iron 0 B r X-Arg-A sp-Tyr-NHNH-Y (I) l 1'X-Asp-Tyr-Met-Gly-Trp-Met-Asp-P llQ-NH: (VI) X-Ser 1 10, 0B. l

l Arg-Asp-Tyr-NHNH-Y (II) S 0 :H 113 Asp-Xyr-Met-Oly-Trp-Met-Asp-Phe-NHz (VIII) X-Ser-NHNH-Y (X) Oli- NOg 0B,

l X-A sp-Arg-A sp-Tyr-NHNH-Y (III 1' I S er-NHNH-Y (XI)X-Asp-Arg-Aspflyr-NHNH: (IV) B. l X-Ileud er-NHNH-Y (XII)X-Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NHz (VII X-Ileu-Ser-NHNH;(XIII) $0 311 Asp-Arg-Asp-Tyr-Met- Gly-Trp-Met-AspPhe-NH: (IX)X-Ileu-Ser-Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH: (XIV) SOaHIleu-Ser-Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH, (XV) Two of thestarting materials, namely, tertiary butyloxycarbonyl-O-benzyl-L-serineand tertiary butyloxycarbonyl-B-benzyl-L-aspartyl-tyroainebenzyloxycarbonyl-hydrazide are well known in the art. The startingmaterial L methionyl-glycyl-L-tryptophyl-L-methionyl-L-aspartyl-L-phenylalanine amide may be prepared by reactingglycyl-L-tryptophyl-L-methionyl-L-aspartyl-L phenylalanine amidetrifluoroacetate with tertiary butyloxycarbonyl-L-methionyl-2,4,S-trichlorophenyl ester. The peptide sequences indicatedin the above reaction scheme may be joined by any known coupling methodof peptide synthesis to form the indicated polypeptides as shown.Partial sequences are first formed by joining together the amino acidsone at a time and then joining the resulting sequences one with anotherto obtain the desired polypeptide product.

Alternatively, the peptides of this invention may be prepared by thesequential addition of the appropriate amino acids one at a time to theL-methionyl-glycyl-L- tryptophyl L methionyl-L-aspartyl L phenylalanineamide.

Such additions are accomplished, for example, by activating thecarboxylic acid group in the amino acid to be added after protecting theamino group in such amino acid, for instance, by converting it to itstertiary-butyloxycarbonyl derivative, converting this derivative into,for example, a nitrophenyl ester and then interacting this active esterwith another amino acid or peptide as desired.

Among the suitable activating groups to be employed in the above processmay be mentioned any group which causes the acid function to become morereactive, such as mixed anhydrides (which normally involves theacylation of an amine with the mixed anhydrides of, for instance, anacyl amino acid and isovaleric acid), azides, acid chlorides, reactionproducts with carbodiimides, reactive N-acyl compounds, O-acylhydroxylamine derivatives, and active esters, such as alkyl esters withelectron attracting (negative) substituents, vinyl esters, enol esters,phenyl esters, halophenyl esters, thiophenyl esters, nitrophenyl esters,2,4-dinitrophenyl esters, and nitrophenylthiol esters.

In forming the peptide sequences in the above manner, the hydroxylprotecting group employed may be benzyl, tertiary butyl,tetrahydropyranyl, and the like, the carboxyl protecting groups may bemethyl, ethyl, tertiary butyl, benzyl, and the like, the guanidineprotecting groups may be nitro, tosyl, p-nitrobenzyloxycarbonyl,protonation, and the like, and the amino protecting groups (X and Y inthe above formulae) may be benzyloxycarbonyl, t-butyloxycarbonyl,trifiuoroacetyl or o-nitrophenylsulfenyl, except that t-butyloxycarbonylmay not be employed in combination with o-nitrophenylsulfenyl.

It should be noted at this point that since protecting group X in theabove formulae must be selectively re-.

moved in order to add the next amino acid in the sequence, it isrequired that X and Q be different.

Various methods of removing protecting groups X and Y from the peptideclaim are known in the art. However, since selective removal of X isrequired, the method chosen will depend upon the protecting groupemployed. For instance, where X represents t-butyloxycarbonyl, it may beremoved by acid treatment, as by trifluoroacetic acid, or the like.Where X represents benzyloxycarbonyl, the removal may be byhydrogenolysis, for instance, in the presence of a palladium on charcoalcatalyst. In those instances where X represents trifiuoroacetyl, it maybe removed by treatment with a nucleophile (e.g., hydrazine in methanol,sodium hydroxide in methanol or an alkoxide such as sodium methoxide orsodium ethoxide, and the like). If X is o-nitrophenylsulfenyl, it may beremoved either by treatment with mild acid [e.g., a hydrohaloacid (suchas hydrobromic or hydrochloric acids) in a solvent such as ethylacetate, ether, or other alkyl ester or a y ether 9lYI1lL or with asulfur nucleoph l such as sodium thiophenoxide, nitrothiophenoxide, andthe like. By proceeding in this manner, selective removal of the Xprotecting group is achieved. Selective removal of Y to form theprotected peptide hydrazide intermediate may be accomplished in the samemanner as set forth above, the appropriate method of removal beingselected, dependent upon the protecting group employed.

The hydroxyl, carboxyl and guanidino protecting groups may be removed byknown reactions, such as hydrogenolysis, treatment with acids such ashydrochloric acid, hydrobromic acid, triuuoroacetic acid, and the like,treatment with alkali, such as sodium or potassium hydroxide, and thelike, or by treatment with sodium in liquid ammonia.

The sulfation of the peptides of this invention containing tyrosine maybe achieved by the reaction of the peptide with concentrated sulfuricacid in the cold for a short period of time. We have discovered,however, that sulfation is greatly facilitated and a significantincrease in yield of the desired peptide ester is obtained if potassiumbisulfite is added to the concentrated sulfuric acid (for example, in aratio of from about 5 to 20 moles of bisulfate to moles of peptide). Thereaction is conducted for a period of time of about 3 to 8 hours attemperatures below 20 C., preferably below 0 C.

The invention will be further illustrated in the following examples. Alltemperatures are in degrees centigrade unless otherwise stated:

EMMPLE 1 L-methionylglycyl-L-tryptophyl-L-methionyl-L-aspartyl-L-phenylalanine amide (A) Tert.butyloxycarbonyl-L-methionylglycyl-L-tryptophyl Lmethionyl-L-aspartyl-L-phenylalanine amide.Glycyl-L-tryptophyl-L-methionyl L aspartyl-L-phenylalanine amidetrifluoroacetate (3.8 g.) is dissolved in a mixture of dimethylformamide(45 ml.) and triethylamine (1.4 ml.) andtert.-butyloxycarbonyl-L-methionine 2,4,5-trichlorophenyl ester (2.5 g.)added. After stirring for three hours at room temperature, the reactionmixture is diluted with ethyl acetate and the precipitate is filtered,washed with ethyl acetate and ether and dried. Yield is 3.4 g.; M.P.-182".

(B) The protected hexapeptide of (A) (3.1 g.) is dissolved in the coldtrifluoroacetic acid (20 m1.) and the solution kept under nitrogen atroom temperature for 25 minutes. Addition of ether (250 ml.)precipitates the trifiuoroacetate which is filtered, washed with etherand dried.

EXAMPLE 2 Tert-butyloxycarbonylnitro-L-arginyl-B-benzyl-L-aspartyl-L-tyrosine benzyloxycarbonylhydrazide (I) fl-Benzyl-L-aspartylL-tyrosine benzyloxyearbonylhydrazide tri-fluoroacetate (1.25 g.) wasdissolved in dimethylformamide (5 ml.) and the solution was cooled in anice-bath. Tri-ethylamine (0.28 ml.) andtert.butyloxycarbonylnitro-L-arginine N-hydroxysuccinimide ester (915mg.) were added. The reaction mixture was stored at room temperature andthree more portions (83 mg. each) of active ester were added after 2,3and 6 hours. After a total reaction time of 24 hours, ethyl acetate (200ml.) was added and the solution was washed with 20% aqueous citric acidand water. After drying (MgSO the solvent was removed in vacuo and theresidue crystallized from ethyl acetate ether. Yield 1.31 g.

EXAMPLE 3 Nitro-L-arginyl-fl-benzyl-L-aspartyl-L-tyrosinebenzyloxycarbonyl-hydrazide trifluoroacetate (II) I (1.11 g.) wasdissolved in trifiuoroacetic acid (7 ml.) and the solution was kept atroom temperature for 15 minutes. The solvent was removed in vacuo andthe residue was titurated with ether. Yield 1.04 g.

EXAMPLE 4 Tert. butyloxycarbony p benzyl L aspartyl-nitro-L- arginyl ,6benzyl-L-aspartyl-Irtyrosine benzyloxycarbonyl-hydrazide (III)Triethylamine (0.17 ml.) was added to an ice-cold solution of III (1.04g.) in dimethylformamide (5 ml.) followed bytert-butyloxycarbonyl-B-benzyl-L-aspartic acid p-nitrophenyl ester (650mg). After 3 hours standing at room temperature, the mixture was dilutedwith ethyl acetate (100 ml.) and the solution was washed with 20%aqueous citric acid and water. After drying (MgSO the solvent wasremoved in vacuo and the residue triturated with a mixture of ethylacetate-hexane. Yield 1.08 g.

EXAMPLE 5 Tert.-butyloxycarbonyl)-Lraspartyl-L-arginyl-L-aspartyl-L-tyrosine hydrazide (IV) A solution of III (900 mg.) in a mixture ofmethanol, acetic acid, water (2: 1 :1) was hydrogenated for 24 hoursover palladium on charcoal (150 mg.). The catalyst was filtered and thefiltrate was concentrated to dryness. The residue was disintegrated withethanol and filtered. Yield 402 mg.

EXAMPLE 6 Tert.-butyloxycarbonyl-L-aspartyl-L-tyrosine hydrazide (V) Asolution of tert. butyloxycarbonyl B-benzyl-L- aspartyl-L-tyrosinebenzyloxycarbonylhydrazide (5 g.) in a mixture of methanol, acetic acidand water (2:1:1) was hydrogenated for 4.5 hours over 10% palladium oncharcoal (800 mg). The catalyst was filtered and the filtrate wasconcentrated to dryness in vacuo. The residue was triturated with ethylacetate. Yield 3.1 g.

EXAMPLE 7 Tert. butyloxycarbonyl-L-aspartyl-L-tyrosyl-L-methionylglycylL tryptophyl L methionyl-L-aspartyl-L- phenyl-alanine amide (VI)Concentrated hydrochloric acid (0.12 ml.) was added to a solution of V(100 mg.) in dimethylformamide (4 ml.), cooled in a dry ice-acetone bathat 20. The temperature of the bath was allowed to rise to and an aqueous14% sodium nitrite solution (0.125 ml.) was added. After 5 minutes thetemperature of the bath was lowered to 25 and N-ethylpiperidine (0.26ml.) was added, followed by 180 mg. of L-methionyl-glycyl-L- tryptophylL methionyl L aspartyl-L-phenylalanine amide trifluoroacetate dissolvedin dimethylformamide (1 ml.). The reaction mixture was stored at 5 andafter 24 hours a second portion of tert.-butyloxycarbonyl-Laspartyl-L-tyrosine azide (from 40 mg. of V) was added. After another24-hours period at 5, the reaction mixture was poured into 30 ml. ofwater containing 1% acetic acid. The precipitate was dried. Yield 200mg.

EXAMPLE 8 Tert. butyloxycarbonyl L aspartyl L arginyl-L- aspartyl Ltyrosyl L methionyl-glycyl-L-tryptophyl Lmethionyl-L-aspartyD-L-phenylalanine amide Concentrated hydrochloricacid (0.12 ml.) was added to a solution of IV (170 mg.) indimethylformamide (1.2 ml.) and cooled in a Dry Iceacetone bath at Thetemperature of the bath was allowed to rise to 15 and an aqueous 14%sodium nitrile solution (0.125 ml.) was added. After 5 minutes thetemperature of the bath was lowered to and N-ethylpiperine (0.26 ml.)was added, followed by 180 mg. of L-methionyl-glycyl- L tryptophyl Lmethionyl-L-aspartyl-L-phenylalanine amide dissolved indimethylformamide (1 ml.). The reaction mixture was stored at 5 for 24hours and a second portion of the tetrapeptide azide (from 68 mg. of IV)was added. After 24 hours the reaction was poured 6 into 30 ml. ofwater. The precipitate was filtered and dried. Yield 250 mg. EXAMPLE 9 Laspartyl O sulfate-tyrosyl L-methiony)-glycyl-L- tryptophyl L methionylL-aspartyl-L-phenylalanine amide (VIII) (A) A solution of VI (320 mg.)in trifluoroacetic acid (7 ml.) was kept under nitrogen at roomtemperature for 15 minutes. Ether (100 ml.) was added and theprecipitate filtered, washed thoroughly with ether and dried.Thismaterial (280 mg.) was added to concentrated sulfuric acid (20 ml.),cooled at 20. The solution was kept in the Dry Ice-acetone bath at --20for 75 minutes. The sulfuric acid solution was poured into ice-waterml.). The precipitate was centrifuged, resuspended in icewater (30 ml.)and 4 N sodium hydroxide was added until a clear solution was obtained.After reacidification to pH 4 With dilute sulfuric acid, the precipitateformed was centrifuged, washed twice with ice-water and dried. Yield 155mg. Chromatography on DEAE Spehadex (with ammonium carbonate bulfer)yielded the desired octapeptide sulfate ester: 30 mg.

(B) A solution of VI (330 mg.) in trifluoroacetic acid (7 ml.) was keptunder nitrogen at room temperature for 15 minutes. Ether ml.) was addedand the precipitate was filtered, washed thoroughly with ether anddried. This material (300 mg.) was added in portions to concentratedsulfuric acid (18 ml.) cooled at 20 with vigorous stirring. After 15minutes a solution of potassium bisulfate in concentrated sulfuric acid(408 mg. in 3 ml.) was added. The reaction mixture was stirred for 75minutes at l5 and then stored at --7 for 285 minutes. The sulfuric acidsolution was poured into cold ether (400 ml.); precipitate was filtered,washed with cold ether, and suspended in cold water. Complete solutionwas then achieved by careful addition of 2 N sodium hydroxide.Acidification with N hydrochloric acid led to the precipitation of thedesired octapeptide sulfate ester. Yield 200 mg.

EXAMPLE 10 L-aspartyl-L-arginyl-L-aspartyl O sulfate-L-tyrosyl-L-methionyl-glycol L tryptophyl L methionyl-L- aspartyl-L-phenylalanineamide (IX) (A) A solution of VH (250 mg.) in trifluoroacetic acid (6ml.) was kept at room temperature under nitrogen for 15 minutes. Ether(100 ml.) was added and the precipitate was centrifuged and washed withether. Yield 212 mg. This material was dissolved in concentratedsulfuric acid (20 ml.) which had been pre-cooled in a Dry Ice-acetonebath at -20. After one hour standing in the cooling bath, the sulfuricacid solution was poured into ice-water (100' ml.) and the precipitateformed was separated by centrifugation, and redissolved in water (30ml.) by careful addition of a 4 N sodium hydroxide. After acidificationto pH 5 with dilute sulfuric acid, the precipitate was separated bycentrifugation, washed with water and dried. Yield mg. The desireddecapeptide sulfate ester was isolated by chromatography on DEAESephadex (with ammonium carbonate buffer). Yield 32 mg.

(B) Following the procedure of Example 9(B), but substituting anequivalent amount of compound VII for the compound VI therein employed,there is obtained the desired decapeptide sulfate in good yield.

EXAMPLE 11 Tert.-butyloxycarbonyl-O-benzyl-L-serinebenzyloxycarbonylhydrazide (X) A solution often-butyloxycarbonyl-O-benzyl-L-serine (3 g.) andbenzyloxycarbonylhydrazine (1.7 g.) in dichloromethane (2 0 ml.) wascooled in an ice-Water bath. Dicyclohexylcarbodiimide (2.06 g.) wasadded and the mixture was stirred 2 hrs. in the cold bath and 4 hrs. atroom temperature. The precipitate formed was filtered and the filtratewas washed with 20% citric acid, water,

saturated sodium bicarbonate and water. After drying (MgSO the solventwas removed in vacuo to yield an oily residue: 4.3 g.

EXAMPLE 12 O-benzyl-L-serine-benzyloxycarbonylhydrazide trifluoroacetate(XI) A solution of X (4.3 g.) in trifluoroacetic acid (20 ml.) was keptat room temperature for 15 minutes. The trifluoroacetic acid was removedin vacuo and the residue was crystallized from ether-hexane. Yield 3.9g.

EXAMPLE 13Tert.-butyloxycarbonyl-L-isoleucyl-O-benz.yl-L-serinebenzyloxycarbonylhydrazide(XII) Triethylamine (1.4 ml.) was added to an ice-cold solution of XI(4.5 g.) in dimethylformamide (20- ml.), followed bytert.-butyloxycarbonyl-L-isoleucine N-hydroxysuccinimide ester (4.18g.). The reaction mixture was kept for 6 hours at room temperature anddiluted with ethyl acetate (200 ml.). This solution was washed with 20%citric acid, water, saturated sodium bicarbonate and water. After drying(MgSO the solvent was removed in vacuo and the residue wasrecrystallized from etherhexane. Yield 5.2 g.

EXAMPLE 14 Tert.-butyloxycarbonyl-L-isoleucyl-serine hydrazide (XIII) Asolution of XII (2.8 g.) in a mixture of methanol, acetic acid and water(2:1:1) was hydrogenated for 6 hours over palladium on charcoal (280mg.) The catalyst was filtered, the filtrate was concentrated to drynessand the residue was crystallized from ether. Yield 1.5 g.

EXAMPLE 15 L-isoleucyl-L-seryl L aspartyl-L-arginyl-L-aspartyl-O-sulfate-L-tyrosyl L methionyl-glycyl-L-tryptophyl-L-methionyl-L-aspartyl-L-phenylalanine amide (XV) Concentratedhydrochloric acid (0.24 ml.) was added to a solution of XIII (170 mg.)in dimethylformamide (1 ml.) cooled in a Dry Ice-acetone bath at -20.The temperature of the bath was allowed to rise to 15 and an aqueous 14%solution of sodium nitrite (0.25 ml.) was added. After minutes thetemperature was lowered to 25 and N-ethylpiperidine (0.6 ml.) was added,followed by a solution of IX (500 mg.) in dimethylformamide (2 ml.).After 24 hours standing at 5, the reaction mixture was concentrated todryness and the residue (XIV) dissolved in trifluoroacetic acid (3 ml.).The solution was kept under nitrogen at room temperature for 15 minutes.Ether was added and the precipitate was filtered and washed thoroughlywith ether. Chromatography on DEAE Sephadex (ammonium carbonate buffer)yielded the desired dodecapeptide sulfate ester: 300 mg.

EXAMPLE 16 Tert.-butyloxycarbonyl-L-arginyl-L-aspartyl-L- tyrosinehydrazide (XVI) A solution of I (300 mg.) in a mixture of methanolaceticacid-water (2:1:1) was hydrogenated for 24 hours over palladium oncharcoal (50 mg.). The catalyst was filtered and the filtrate wasconcentrated to dryness. The residue was disintegrated with ethylacetate and filtered. Yield 150 mg.

EXAMPLE 17 Tert.-butyloxycarbonyl L arginyl-L-aspartyl-L-tyrosyl-L-methionyl-glycyl L tryptophyl L methionyl-L- aspartyl-L-phenylalanineamide (XVII) Concentrated hydrochloric acid (0.12 ml.) was added to asolution of XVI (138 mg.) in dimethylformamide (1.2 ml.) and cooled in aDry Ice-acetone bath at -20. The tem erature of the bath was allowed torise to and an aqueous 14% sodium nitrite solution (0.125 ml.) wasadded. After 5 minutes the temperature of the bath was lowered to -25and N-ethylpiperidine (0.26 ml.) was added, followed by 180 mg. ofL-methionyl-glycyl- L-tryptophyl-L-methionyl-L-aspartyl L phenylalamineamide dissolved in dimethylformamide (1 ml.). The reaction mixture wasstored at 5 for 24 hours and a second portion of the tetrapeptide azide(from 51 mg. of XV) was added. After 24- hours the reaction was pouredinto 30 ml. of water. The precipitate was filtered and dried. Yield 235mg.

EXAMPLE 18 L-arginyl-L-aspartyl Osulfate-L-tyrosyl-L-methionylglycyl-L-tryptophyl L methionyl Laspartyl-L- phenylalanine amide (XVIII) Following the procedure ofExample 9, but substituting 235 mg. of XVII for VI, there is obtainedthe above. Yield 140 mg.

EXAMPLE 19 Tert.-butyloxycarbonyl L seryl-L-aspartyl-L-arginyl-L-aspartyl-O-sulfate-L-tyrosyl L methionyl-glycol-L-tryptophyl-L-methionyl-L-aspartyl L phenylalanine amide (XIX)Concentrated hydrochloric acid (0.24 ml.) was added to a solution oftert.-butyloxycarbonyl-L-serine hydrazide (115 mg.) in dimethylformamide(1 ml.) cooled in a Dry Ice-acetone bath at -20. The temperature of thebath was allowed to rise to 15 and an aqueous 14% solution of sodiumnitrite (0.25 ml.) was added. After 5 minutes the temperature waslowered to 25 and N- ethylpiperidine (0.6 ml.) was added, followed by asolution of IX (500 mg.) in dimethylformamide (2 ml.). After 24 hoursstanding at 5 the reaction mixture was concentrated to dryness and theresidue was tn'turated with water and then filtered and washed withethanol. Yield 290 mg.

EXAMPLE 20 L-seryl L aspartyl-L-arginyl-L-aspartyl-O-sulfate-L- tyrosylL methionyl-glycyl -L- tryptophyl-L-methionyl-L-aspartyl-L-phenylalanineamide XIX (290 mg.) was dissolved in trifluoroacetic acid (3 ml.) andthe solution was kept at room temperature under nitrogen for 15 minutes.Ether was added ml.) and the precipitate was filtered, washed with etherand dried. Chromatography on DEAE Sephadex yielded the desiredhendecapeptide sulfate ester. Yield 200 mg.

EXAMPLE 21 O-sulfate-L-tyrosyl-L-methionyl-glycyl-L-tryptophyl-L'methionyl-L-aspartyl-L-phenylalanine amide Following the procedure ofExample 9, but substituting an equivalent amount ofL-tyrosyl-L-methionyl-glycyl-b methionyl-L-aspartyl-L-phenylalanineamide for VI, there is prepared the above.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

What is claimed is:

1. A method of sulfating the tyrosyl moiety of a peptide of the formulaR-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH and pharmaceutically acceptablesalts thereof wherein R is wherein X is benzyloxycarbonyl,t-butyloxycarbonyl, trifiuoroacetyl or o-nitrophenylsulfenyl, whichcomprises contacting the peptide with a mixture of potassium bisulfateand concentrated sulfuric acid, the molar ratio of bisulfate to peptidebeing from about -20: 1, for a period of up to 8 hours at a temperaturebelow 20 C.

2. A method according to claim 1 wherein the peptide has the formulaR-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH and pharmaceutically-acceptableacid addition and ammonium salts thereof wherein R is L-tyrosyl,L-aspartyl-L-tyrosyl, X-L-arginyl-L-aspartyl-L-tyrosyl,L-arginyl-L-aspartyl-L-tyrosyl,L-aspartyl-L-arginyl-L-aspartyl-L-tyrosyl,X-L-aspartyl-L-arginyl-L-aspartyl-L-tyrosyl,L-seryl-L-aspartyl-L-arginyl-L-aspartyl-L-tyrosyl,X-L-seryl-L-aspartyl-L-arginyl-L-aspartyl-L-tyrosyl,L-isoleucyl-L-seryl-L-aspartyl-L-arginyl-L-aspartyk L-tyrosyl, orX-L-isoleucyl-L-seryl-L-aspartyl-L-arginyl-L-aspartyl- L-tyrosyl,

wherein X is benzyloxycarbonyl, t-butyloxycarbonyl, trifluoroacetyl oro-nitrophenylsulfenyl 3. A method according to claim 1 wherein R isL-Tyr, L-Asp-L-Tyr, or L-Ile-L-Ser-L-Asp-L-Arg-L-Asp-LIyr.

4. A method according to claim 1 wherein the reaction takes place at atemperature of 20" C. to 7 C.

5. A method according to claim 1 wherein the peptide is added in portionto concentrated sulfuric acid cooled at -20 C. with vigorous stirring,after 15 minutes a solution of potassium bisulfate in concentratedsulfuric acid is added and the mixture stirred for 75 minutes at -'15 C.and then stored at 7 C. for 285 minutes.

6. A method according to claim 5 wherein the peptide has the formulaR-L-Met-Gly-L-Trp-L-Met-L-Asp-L-Phe-NH 10 andpharmaceutically-acceptable acid addition and ammonium salts thereofwherein R is L-tyrosyl, L-aspartyl-L-tyrosyl,X-L-arginyl-L-aspartyl-L-tyrosyl, L-arginyl-L-aspartyl-L-tyrosyl,L-aspartyl-L-arginyl-L-aspartyl-L-tyrosyl,X-L-aspartyl-L-arginyl-L-aspartyl-L-tyrosyl,L-seryl-L-aspartyl-L-arginyl-L-aspartyl-L-tyrosyl,X-L-seryl-L-asparty1-L-arginy1-Laspartyl-L-tyrosyl,L-isoleucyl-Lseryl-L-aspartyl-L-arginyl-L-aspartyl-L- tyrosyl, orX-L-isoleucyl-L-seryl-L-aspartyl-L-arginyl-L-aspartyl-L- tyrosyl,

References Cited UNITED STATES PATENTS 2,174,131 9/1939 Lubs 260-4572,970,165 1/ 1961 Michel et a1. 260-457 3,579,494 5/1971 Ondetti et a1.260-1125 OTHER REFERENCES Ondetti et al., J. Am. Chem. Soc. 92, -197(1970). Dodgson et al., Biochem. Jr. 71, 10 (1959). Tallan et al., J.Biol. Chem. 217, 703-704 (1955). Reitz et al., J. Am. Chem. Soc. 68,1024-1031 (1946).

ELBERT L. ROBERTS, Primary Examiner g;;g UNITED STATES PATENT @EETEECERTIFICATE m RRECTIN Patent No. 3, Dated December 1].,

Inventor(s) Miguel A. Ondetti et a1.

It is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

Column 2, formula (VIII), 3 should read W 3 Asp-Xyr-Met-Oly--Asp-TyrMet-Gly Column 3, line 3, "tyroaine" should read tyrosine--.

Column 3, line 46, "guanidine" should read -guanidino. Column 4, line35, "Olycyl" should read --Glycyl Column 5, line 2, "butyloxycarbony"should read butyloxycarbonyl-. Column 5, line 6, "III" should read-II--.

Column 5, line 54, "hours" should read hour-.

Column 5, line 61, (VVI)" should read (VII)-.

Column 6, line 4, "methiony" should read methionyl.

Column 6, line 20, "Spehadex" should read -Sephadex--.

Column 6, line 69, "ter." should read -tert.-. Column 8, line 22,"glycol" should read glycyl-.

Column 8, line 55, after "glycyl-L-" insert --tryptophylL Signed andsealed this 21st day of May 197A;

(SEAL) Attest:

EDWARD 1"I.l-"LET-31--IER,JZ2. MITRSIIALL DAL'IJ Attesting Officeriiommlssloner of Patents

